The ability to determine the sex of an embryo soon after fertilization would provide numerous advantages in the livestock and dairy industries as well as in veterinary medicine. In the dairy and beef cattle industry advances in embryo transfer has resulted in a great demand for a method of quickly determining the sex of embryos and cells taken from embryos at early stages of development. The commercial efficiency of livestock and dairy operations would be greatly improved by allowing gestation to be established with embryos of the desired sex. Given the advantage to the dairy industry of a preponderance of female progeny, it would be advantageous if the sex of embryos could be routinely determined prior to embryo transplant into a maternal host. The advantages of sexed embryos are numerous including the selection of replacement of stock based on desired characteristics, such as size, weight, increased milk production, etc. In addition, certain diseases, such as X-chromosome-linked diseases in humans and similar diseases in other mammals, affect individuals of only one sex. Early determination of the sex of an embryo which, if carried to term, would likely be an individual with such a disease would be particularly advantageous and provide valuable information on which to base a decision to allow further development.
Efficient determination of the sex of a conceptus in vivo is also of significant economic importance, and would have important commercial applications. In the dairy and livestock industries, in pregnancies which arise via artificial insemination or natural mating, early determination of the sex of an embryo or fetus would allow for termination of the pregnancy if an embryo or fetus of the desired sex was not obtained.
In situations where, for health or economic reasons, a determination of the sex of an embryo or fetus is indicated, it is important to determine the sex as soon as possible after fertilization. There is a substantial increase in risk to the life and health of a female if abortion is induced late in gestation. With livestock, it is commercially inefficient, both because of reduced reproductive efficiency and dangers to the life and health of the female, to carry an embryo longer than necessary.
With advances in reproductive biology, it would be feasible to avoid all risks and costs associated with pregnancy and abortion if it were possible to determine the sex of an embryo, whether produced in vivo or in vitro prior to or at the time of transfer, and also to determine as early as possible with certainty the sex of an embryo or fetus in vivo.
The sex of a mammal is determined by the presence or absence of the entire Y-chromosome or some functional portion thereof. Genes present on the Y-chromosome govern formation and the development of the male phenotype. The sex of an individual mammal is therefore dependent upon whether or not its genome contains particular DNA sequences, especially those sequences comprising that part of the Y-chromosome which encode genes responsible for sex determination.
The sex or presumptive sex of a mammal can therefore be determined by analysis for Y-specific genes in the DNA of the individual mammal. Alternatively, sex can be determined by unrelated but genetically linked sequences which are associated specifically with the Y-chromosome, preferably on sequences linked closely to the male-determining genes to reduce possible errors in analysis due to genetic recombination.
Prior to the present invention a number of investigators have identified DNA sequences which hybridize preferentially or exclusively to male DNA. See Kunkel et al., Science 191, 1189-1190 (1976); Bishop et al., Nature 303, 831 (1983); Vergnaud et al., Brit. Med. J. 289, 73-76 (1984); Lau et al., The Lancet, Jan. 7, 1984, pp 14-16; Gosden et al., The Lancet, Dec. 25, 1982, pp 1416-1419; Bostick et al., Nature 272, 324 (1978). These DNA sequences have not been functionally characterized, and it is unknown whether these sequences are capable of hybridization to non-human species.
The isolation of sperm separated according to the sex chromosome they contain, and using these sperm to fertilize ova is one method currently used to control embryo-sex. Such sperm isolation methods are significantly limited due to the difficulty of obtaining preparations of sperm in which more than 99% of the sperm carry the sex chromosome of only one of the sexes. At present, known techniques for separating sperm according to sex are not practical for obtaining mixtures of sperm with more than about 75% harboring the same sex chromosome. Therefore determining sex by segregating sperm is limited by economic and commercial considerations.
Karyotyping fetal cells obtained after several weeks gestation by amniocentesis, chorion biopsy, and other procedures is another known method of determining sex. Such procedures are limited, however, in commercial application due to the expense, risk of infection, and time required to carry out this type of analysis.
Other prior art attempts to deal with this problem have been indirect and incomplete. U.S. Pat. No. 4,769,319 issued to Ellis et al. discloses male specific nucleic acid hybridization probes which have sequences complementary to sequences of segments in bovine male specific DNA. These nucleic acid sequences are stated to be useful as hybridization probes for sexing embryos and fetuses. Australian Patent Application No. 59561/86 discloses bovine DNA probes which hybridize preferentially to male DNA, and are also stated to be useful in sexing embryos and fetuses. These DNA sequences are indicated to be species specific. International Patent Application No. PCT/AU87/00254, discloses a 307 base pair nucleic acid sequence designated BRY.1 comprising Y-specific DNA which is capable of hybridizing with male bovine and ovine derived DNA but not with DNA isolated from female animals. PCT Application No. PCT/AU 89/0029 discloses nucleic acid isolates capable of hybridizing to Y-specific DNA sequences of ruminants.
Moreover, there is nothing in the prior art to indicate that any like DNA segments exist which could be used to provide the basis of a polynucleotide probe to sex by nucleic acid hybridization, with as few as 2 cells, with virtually 100% accuracy, in an extremely short time period, a mammalian embryo at a morula or blastocyst stage, at or before transfer of the embryo for further development.